Efficient discrimination against RNA-containing primers by human DNA polymerase ε

DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and catalyzes a covalent attachment of dNMP to the 3' end of the primer. Previous studies have shown that Polε easily inserts and extends ribonucleotides, which may promote mutagenesis and genome instability. In this work, we analyzed the mechanisms of discrimination against RNA-containing primers by human Polε (hPolε), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPolεCD extends RNA primers with approximately 3300-fold lower efficiency in comparison to DNA, and addition of one dNMP to the 3′ end of an RNA primer increases activity 36-fold. Likewise, addition of one rNMP to the 3′ end of a DNA primer reduces activity 38-fold. The binding studies conducted in the presence of 0.15 M NaCl revealed that human hPolεCD has low affinity to DNA (KD of 1.5 µM). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces the stability of the hPolεCD/DNA complex by 25-fold. Upon template:primer binding, the incoming dNTP and magnesium ions make hPolε discriminative against RNA and chimeric RNA–DNA primers. In summary, our studies revealed that hPolε discrimination against RNA-containing primers is based on the following factors: incoming dNTP, magnesium ions, a steric gate for the primer 2′OH, and the rigid template:primer binding pocket near the catalytic site. In addition, we showed the importance of conducting functional studies at near-physiological salt concentration.

www.nature.com/scientificreports/ this work, we performed functional studies of hPolε at near-physiological salt concentration by employing presteady-state and binding kinetics. We studied the discrimination of hPolε against RNA-containing primers at template:primer binding and dNTP incorporation steps. In addition, we examined the effect of dNTP, magnesium ions, and salt concentration on the stability of hPolε/template:primer complexes.

Results
Salt concentration has a dramatic effect on the stability of the hPolε/DNA complex. The catalytic domain of human DNA polymerase ε (hPolε CD ; residues 27-1172) with inactivated exonuclease activity was expressed in Escherichia coli and purified to near homogeneity (Suppl. Figure S1). The concentrated sample had a brownish color due to the presence of a [4Fe-4S] cluster 21,22 . Before starting functional studies with the obtained hPolε CD sample, we estimated the level of molecules possessing DNA binding activity. Increasing amounts of protein were added to reactions containing 0.5 µM DNA, and the level of DNA in complex with a protein was estimated by electrophoretic mobility shift assay (EMSA; Fig. 1). It was found that a hPolε CD sample with homogeneity greater than 90% (Suppl. Figure S1) has a level of active molecules of 55.4 ± 4.3%. All data described below were obtained using the active enzyme concentration. The analysis of Polε CD interaction with DNA (Suppl. Table S1) was performed at near-physiological salt concentration using an Octet K2, which employs Bio-Layer Interferometry (BLI) technology to monitor molecular interactions in real time. This approach allows obtaining the rate constants of complex formation (k on ) and dissociation (k off ) as well as the dissociation constant (K D ). A 23-mer DNA template with biotin at the 5′-end was primed by an 11-mer DNA primer and loaded on a streptavidin-coated sensor. Primer length selection was based on structural data showing that ten base-pairs in a duplex is sufficient to form all necessary contacts with the conserved catalytic domain of yeast Polε CD 23, 24 . Binding studies revealed that in the presence of 0.15 M NaCl hPolε CD binds a DNA duplex with low affinity (K D = 1.5 µM, Table 1). The obtained k off value of 0.46 s −1 indicates that the half-life of the hPolε CD /DNA complex is ~ 1.5 s on average. Surprisingly, previous reports showed one to two orders of magnitude stronger interaction   16,18 . For yeast Polε and Polε CD , the corresponding K D values are 11.6 nM and 15.6 nM, respectively 19 . The discrepancy with our data might be explained by the absence of salt in those studies. Of note, the obtained k on value of 304 mM −1 s −1 (Table 1) is close to the previously reported k on value of 270 mM −1 s −1 acquired by a different approach based on the measurement of DNA polymerase activity 16 .
To analyze the effect of salt concentration on the stability of the hPolε CD /DNA complex, we also conducted binding experiments in the presence of 0.1 M NaCl. Strikingly, a decrease in salt concentration from 0.15 M to 0.1 M reduced K D by 25-fold (from 1.52 µM to 61 nM), which is mainly due to the 21-fold reduction in k off value (Table 1). Consistently, in the presence of 125 mM NaCl, the hPolε/DNA complex showed a K D of 532 nM, which is 8.7-fold higher than the K D value obtained at 100 mM NaCl. The dramatic effect of the ionic strength on the stability of the hPolε CD /DNA complex will be discussed below.
We analyzed how the incoming dNTP at near-physiological salt concentration 25 affects the interaction of hPolε CD with DNA. DNA polymerases use divalent metals to coordinate the triphosphate moiety of dNTP, so magnesium chloride was added to the reaction as well. Addition of 50 µM dTTP and 5 mM MgCl 2 in the presence of 0.15 M NaCl resulted in a 2.5-fold reduction in affinity of hPolε CD to DNA, with an almost two-fold increase in k off value (Table 1). A similar effect was observed at 0.1 M NaCl, with K D and k off being increased by two-and 1.5-fold, respectively. The following exploration revealed that Mg 2+ ions make a major impact on Polε interaction with a template:primer (see below).
The incoming dNTP and Mg 2+ aid hPolε in discrimination against RNA and chimeric primers upon template:primer binding. We analyzed how hPolε CD discriminates against RNA and chimeric RNA-DNA primers upon binding them in the presence and absence of dTTP/Mg 2+ . Without them, similar K D values were obtained for all types of primers ( Table 2; Fig. 2). The addition of 50 µM dTTP and 5 mM MgCl 2 reduced the stability of all complexes but to varying extents depending on primer structure. For a chimeric primer with seven dNMPs at the 3′ end, K D increased only 1.5-fold, which is close to the two-fold increase observed for the DNA primer. In the presence of dTTP/Mg 2+ , the chimeric primers with one or three dNMPs at the 3′ end demonstrate K D values that are 3.5-fold higher compared to the DNA primer ( Table 2; Fig. 2). In comparison, the discrimination factor reduced ~ two-fold for a primer with five dNMPs at the 3′ end.
In order to estimate how insertion of one rNMP affects interaction of hPolε with the template:primer, we used a DNA primer with a 3′-deoxy-cytosine. This primer has a 2′-hydroxyl to imitate ribonucleotide and lacks 3′OH to prevent primer extension. Upon addition of dTTP/Mg 2+ , the affinity of hPolε CD to the template:primer decreased five-fold, which is similar to the 5.8-fold reduction observed for an RNA primer with a dideoxy-cytosine at the 3′-end (Table 2; Fig. 2). Direct comparison of affinity to a DNA primer and to a primer with 3′-rNMP demonstrates a 1.9-fold higher selectivity for DNA. Therefore, hPolε senses the primer 2′-hydroxyl only in the presence of incoming dNTP and Mg 2+ .  www.nature.com/scientificreports/ The effect of either 5 mM MgCl 2 or 50 µM dTTP on hPolε CD affinity to a template:primer was analyzed using the following primers: DNA, R10-D1, and D10-R1 (Fig. 3). Interestingly, these additives added separately or together had a stronger impact on hPolε interaction with RNA-containing primers. The most impressive effect was observed for R10-D1, where addition of either dTTP or Mg 2+ increased K D values by 1.8-and 7.5-fold, respectively (Fig. 3). Thus, in the presence of dTTP, hPolε has a 1.9-fold higher affinity to a DNA primer versus R10-D1; in the presence of Mg 2+ , the difference is three-fold. Of note, discrimination against both RNA-containing primers is slightly higher in the presence of dTTP/Mg 2+ than Mg 2+ (Fig. 3). These data indicate that, upon ternary complex formation, the incoming dNTP and Mg 2+ help hPolε to discriminate against RNA-containing primers until they are extended with several dNMPs. hPolε extends RNA-containing primers with greatly reduced activity. It was previously shown that Polε is able to incorporate ribonucleotides into a growing DNA strand and to extend them 26,27 . Moreover, Polε can extend R-loops, which present the DNA template primed by RNA as well as a displaced complemen-  Table 2 are presented as bar graphs showing the mean ± SD (n = 3). The primer type is indicated below the bar. Asterisk indicates the absence of primer 3′-hydroxyl. www.nature.com/scientificreports/ tary DNA strand 28 . Accordingly, we decided to explore how the presence of ribonucleotides in a primer affects its extension by hPolε CD by employing the pre-steady-state kinetic approach. Single-nucleotide incorporation experiments were performed under single-turnover conditions by providing a two-fold excess of hPolε CD over DNA (Suppl . Table S1). hPolε CD (0.88 µM) was incubated with a Cy3-labeled DNA (0.4 µM) and quickly mixed with an equal volume of 2 mM dTTP and 10 mM MgCl 2 under rapid chemical quench conditions. The fraction of extended primer was plotted against time ( Fig. 4) and the data were fit to a single-exponential equation (Eq. 1). hPolε CD extends the DNA primer with a rate of 62.4 s −1 obtained at 35 °C in the presence of 0.1 M NaCl (Fig. 4A). In comparison, the catalytic domain of hPolα has a k pol of 33.8 s -1 at conditions similar to those used in this work 29 . These results are in line with the recently reported average rate of replication fork movement in yeast cells, which is ~ 50 nucleotides per second 30 . A significantly higher k pol value of 248 s -1 was obtained for the hPolε CD at 20 °C in the absence of salt 16 . The authors of that study noted that the rate of polymerization was > 500 s −1 at 37 °C. The almost tenfold difference in activity with our data is probably due to the inhibitory effect of salt concentration as previously demonstrated 31 .
Activity of hPolε CD in extension of RNA and chimeric primers was analyzed at the same conditions as with the DNA primer. Strikingly, the rate of RNA primer extension is 3300-fold lower compared to DNA (Fig. 4B). Such a severe effect on catalysis indicates that pre-catalytic positioning of the primer 3′-hydroxyl relative to the α-phosphate of dNTP and/or the catalytic metal is changed. Upon addition of dNMPs to the 3′-end of an RNA primer, the rate of primer extension gradually increases (Fig. 4C-F). Thus, the rates of dNMP incorporation on the chimeric RNA-DNA primers with one, three, five, and seven dNMPs at the 3′-end are 92-, 22-, 3.8-, and 1.6-fold lower, respectively, than on DNA (Fig. 5).
Interestingly, addition of just one dNMP to the 3′-end of an RNA primer increases activity 36-fold, from 0.019 s −1 to 0.682 s −1 , while the subsequent addition of every two dNMPs increases activity only several-fold (Fig. 4). Moreover, addition of one ribonucleotide to the 3′-end of a DNA primer reduces the rate of dTTP incorporation 38-fold, from 62.4 s −1 to 1.63 s −1 (Fig. 6). A similar effect on Polε activity upon addition of either 3′-rNMP to a DNA primer or 3′-dNMP to an RNA primer indicates the presence of a steric gate for 2′OH. Indeed, analysis of the structure of yeast Polε CD in complex with DNA and dNTP 23,24 revealed a conserved Thr876 (Suppl. Figure S2) located under the sugar and sensing the 2′-hydroxyl of a primer (Fig. 7). The other steric gate   This study has shown that hPolε CD exhibits low affinity to the template:primer at physiological salt concentration, with a 19-fold higher K D value than the 79 nM obtained in the absence of salt 16 . Interestingly, hPolα shows a similar with Polε affinity to DNA in the presence of 100 mM (K D = 41 nM) and 150 mM NaCl (K D = 1.4 µM), despite the absence of the so-called processivity domain that is unique to Polε 35 . According to structural data [22][23][24] , this domain makes three to four additional hydrogen bonds with a template:primer (a detailed comparison of hPolα and yPolε interactions with a template:primer are provided in 36 ). In other work, we have shown that hPolε CD with an intact exonuclease active site and without internal truncations also exhibits a strong sensitivity to ionic strength upon template:primer binding 37 .
The results of our binding studies support the idea that PCNA is required for processive synthesis of the leading strand, according to the current model of the eukaryotic replication fork 38 . The recently published structure of yeast Polε holoenzyme revealed that accessory subunits do not interact with the DNA duplex 5 , indicating that the holoenzyme footprint on a DNA duplex is only ten base-pairs, the same as for the catalytic domain 22,24 . Polε is flexibly attached to the CMG helicase through interaction with the C-terminal domain of the catalytic subunit and the N-terminal domain of the second accessory subunit 4,7 . In contrast to PCNA, interaction with CMG does not prevent the Polε catalytic domain from occasional dissociation from the growing primer end.
The main role of Polε is a synthesis of the leading DNA strand, but it may handle RNA and chimeric RNA-DNA strands in certain circumstances. One example is a ribonucleotide at the growing primer end, which Polε can insert and extend 26,27 . Another example is the ability of Polε to extend the 3′-ends of R-loops, serving as a possible way to restart DNA replication 28 . In addition, it is possible that Polα sometimes preliminarily terminates DNA synthesis due to generation of a mismatch, like purine-purine, which is difficult to extend. In this case, Polε may bind the chimeric RNA-DNA primer, proofread the non-cognate nucleotide, and extend the corrected primer with dNMPs until the length of a primer is enough for RFC to load PCNA. A similar mechanism was suggested for the lagging strand where Polδ proofreads the Polα-generated mismatches 39 . Intriguingly, some portion of hPolε is associated with RNA polymerase II independently of the cell cycle 40 . Moreover, according to results of UV cross-linking studies, hPolε is located in close proximity to the newly synthesized RNA strand 40 . These data suggest that Polε could play a currently unknown role in RNA transcript processing.
As we found, hPolε CD shows approximately 3,000-fold lower activity in extension of RNA versus DNA primers, making it unlikely that Polε plays a role in restarting the replication fork from the R-loops. The k obs value of 0.019 s −1 indicates that half of RNA primers will be extended with one dNMP in ~ 40 s. The best candidate Figure 7. Polε has a steric gate for the 2′-hydroxyl of a primer. The protein is represented as cartoon and colored cyan; Thr876 is shown as sticks. The template and primer are shown as sticks and colored blue for nitrogen, red for oxygen, orange for phosphorus, and grey for carbon. The O1 of Thr876 and modeled O2 of primer Cyt are presented as spheres with 60% transparency and colored red. The interception of spheres indicates steric hindrance. The crystal structure of the ternary complex of yeast Polε CD with DNA and dCTP (pdb code 6hiv (22)) was used for this presentation prepared with the PyMOL Molecular Graphics System. www.nature.com/scientificreports/ for R-loop extension would be Polα, which displays a similar rate of DNA and RNA primer extension 29 . On the other hand, upon extension of an RNA primer with dNMPs, hPolε CD activity gradually increases, and the chimeric primer with seven dNMPs is extended fairly well (Fig. 4). Our studies have shown that, in comparison to template:primer binding, the rate of dNMP incorporation is more sensitive to the presence of ribonucleotides in the primer. It is interesting that despite their similarly organized DNA binding sites 29 , Polα and Polε demonstrate such a significant difference in selectivity to a DNA primer upon catalysis of phosphodiester bond formation. Incorporation of ribonucleotides to the nascent DNA strand by replicative DNA polymerases could be a challenge for genome integrity 41,42 . It was reported previously that hPolε CD readily inserts ribonucleotides, and almost half of them escape proofreading by intrinsic exonuclease 27 . Interestingly, activity of hPolε was reduced only two-to three-fold when one-three consecutive ribonucleotides were added to the primer 3′-end 27 . Such a low effect of 3′-rNMPs on primer extension may be due to the absence of salt in reaction and to the type of assay where the rate of enzyme/DNA complex formation limits the reaction rate due to low concentrations of DNA and Polε and the presence of 10% glycerol. Our study, performed at single-turnover conditions and in the presence of 0.1 M NaCl, demonstrates much stronger sugar selectivity, with a 38-fold reduction in activity by 3′-rNMP (Fig. 6). Due to the balance between DNA polymerase and exonuclease activities 43 , the hampered extension of 3′-ribonucleotides will result in increased probability of their excision.
A notable finding of the current study is the role of incoming dNTP and magnesium ions in the discrimination of hPolε against primers containing ribonucleotides. When dNTP and Mg 2+ are not present, hPolε binds DNA, RNA, and chimeric primers with similar affinity. In the presence of dTTP/Mg 2+ , selectivity for DNA primers increases two-to four-fold. Interestingly, Mg 2+ ions play the main role in discrimination against RNA-containing primers upon template:primer binding (Fig. 3). Magnesium ions most likely interact with DNA phosphates and compete with DNA-binding residues of hPolε CD . In the case of RNA-containing primers, a stronger effect of Mg 2+ on the hPolε/template:primer interaction can be due to steric hindrances generated by the wide helix of DNA:RNA duplexes and by the primer 2′-hydroxyl. Of note, dTTP alone has a stronger impact on hPolε affinity to the R10-D1 primer than to DNA (Fig. 3). This is an interesting example of how one substrate modulates enzyme selectivity for another. Noteworthy, there is no discrimination against RNA primers extended with seven or more dNMPs.
Available structural data provide clues as to why, upon template:primer binding, hPolε discriminates against the chimeric primers with one or three dNMPs with similar efficiency and does not discriminate against primers containing seven dNMPs at the 3′ end. Polε interacts with a ten base-pair duplex, where the first four base pairs from the growing primer end interact with the rigid DNA-binding pocket established mainly by the palm subdomain with significant contributions by the main-chain atoms [22][23][24] . The remainder of the template:primer interacts with the flexible thumb, which can adjust its conformation to accommodate a wider DNA:RNA duplex. The absence of discrimination against RNA when dNTP and Mg 2+ are not added may be due to the slightly different mode of duplex binding, which reduces steric hindrance between the wide DNA:RNA duplex and the rigid DNA-binding cleft. dNTP and Mg 2+ binding and the fingers closing likely forces hPolε interaction with the template:primer in a specific way, which results in steric hindrance and reduced affinity to RNA and chimeric primers.

Materials and methods
Cloning, expression, and purification. The catalytic domain of hPolε (hPolε CD ), with the proofreading 3′ → 5′ exonuclease inactivated by the double mutation D275N/E277Q, was cloned into a pASHSUL-1 plasmid 44 to produce the recombinant protein with a cleavable N-terminal His 6 -Sumo tag. To increase protein solubility and yield, we deleted the external loop spanning amino acids 185-209. The corresponding region is distant from the active center and is mostly disordered in all yeast Polε CD structures.
hPolε CD were expressed in E. coli strain Rosetta-2 (DE3) at 18 °C for 16 h following induction with 0.2 µg/ ml anhydrotetracycline. Afterward, the cells were harvested by centrifugation at 4000 g for 15 min, washed with PBS, aliquoted and maintained at − 80 °C.
The purification protocol includes chromatography on a Ni-IDA column (Bio-Rad), His-Sumo-tag digestion during overnight dialysis, and chromatography on a Heparin HP HiTrap column (GE Healthcare) as well as on a size-exclusion column in buffer containing 25 mM Tris-HEPES (pH 7.8), 0.15 M NaCl, 1% glycerol, and 2 mM tris(2-carboxyethyl)phosphine (TCEP). Finally, samples were concentrated to 30-60 µM and flash-frozen in aliquots. Protein concentrations were estimated by measuring the absorbance at 280 nm and using extinction coefficients of 154 mM −1 cm −1 . The extinction coefficients were calculated with ProtParam 45 .
Estimation of the level of DNA-binding (active) molecules by electrophoretic mobility shift assay. Reactions containing 0.5 µM DNA (Suppl . Table S1) and varying amounts of protein were incubated in 10 μl for 5 min at room temperature in the buffer containing 20 mM Tris-Hepes (pH 7.8), 100 mM NaCl, 2% glycerol, 2 mM TCEP, and 0.2 mg/mL BSA; 5 μl was then loaded on 5% native PAGE. Samples labeled with Cy3-dye were visualized using the Typhoon 9410 imager (GE Healthcare) and quantified using ImageJ software (version 1.53 s, National Institutes of Health). The mean value and standard deviation are calculated from three independent experiments.
Binding studies. Analysis of binding kinetics was done at 23 °C on an Octet K2 (Sartorius AG). This device uses Bio-Layer Interferometry technology to monitor molecular interactions in real time. A template with a biotin-TEG at the 5′-end was annealed to the primers (Suppl . Table S1) and immobilized on a streptavidin-coated biosensor (SAX, Sartorius AG). Primers were added at two-fold molar excess over the template. SAX sensors were loaded with oligonucleotide-biotin at 50 nM concentration for 7 min at 500 rpm. Then sensors were blocked by incubating for 2 min in 10 µg/ml biocytin. In the first row of a 96-well microplate (Greiner Bio-One), the first www.nature.com/scientificreports/ six wells contained the buffer, consisting of 30 mM Tris-Hepes, pH 7.8, 100 mM or 150 mM NaCl, 2 mM TCEP, and 0.002% Tween 20. The next six wells contained two-fold dilutions of hPolε CD in the same buffer. All wells in the adjacent row contained only the buffer for reference. Examples of binding curves are shown in Suppl. Figure S3, where the effect of either 5 mM MgCl 2 or 50 µM dTTP on the stability of the hPolε CD /DNA complex was analyzed. Data Analysis HT software (ver. 11.1, Sartorius AG) was used to calculate binding constants (k on , k off , and K D ) by using global fitting. The average value and standard deviation were calculated from three independent experiments.
Kinetic studies. Pre-steady-state kinetic studies were performed on the QFM-4000 rapid chemical quench apparatus (BioLogic, France) at 35 °C. Reactions contained 0.44 µM hPolε CD (active molecules), 0.2 µM DNA, 1 mM dTTP, 25 mM Tris-HEPES, pH 7.8, 0.1 M NaCl, 5 mM MgCl 2 , 2 mM TCEP, and 0.2 mg/mL BSA. hPolε CD was incubated with a Cy3-labeled 15-mer primer annealed to a 25-mer DNA template (Suppl . Table S1), to allow formation of the binary complex, and rapidly mixed with a solution containing 2 mM dTTP and 10 mM MgCl 2 followed by quenching with 0.3 M EDTA. Products were collected in a tube containing 15 µl 100% formamide and separated by 20% Urea-PAGE. The Cy3-labeled products were visualized using the Typhoon FLA 9500 (GE Healthcare) and quantified by ImageJ, version 1.53 (NIH). The extended primer fraction was calculated by dividing the amount of extended primer by the amount of primer added in reaction. The percent of extended primer was plotted against time and the data fit to a single-exponential equation: where A is the amplitude, k obs is the observed rate for dNTP incorporation, and t is the time.

Data availability
The data that support the findings of this study are included in the Supplementary Information file or available from the corresponding author on request.